Production Of γ-aminobutyric Acid In Recombinant Corynebacterium Glutamicm Through Co-expressing Glutamate Decarboxylase Genes From Lactobacillus Brevis

γ-aminobutyric acid (GABA) is synthesized by glutamate decarboxylase (GAD) whichcatalyzes the irreversible α-decarboxylation of L-glutamate. GABA has various physiologicalfunctions and is widely used in food, pharmaceutical, and agricultural fields.In the previous study, a single-step GABA fermentation system by recombinantCorynebacterium glutamicum strain expressing one glutamate decarboxylase gene derivedfrom Lactobacillus brevis Lb85had been created. The recombinant strain can directly convertits own accumulated L-glutamate into GABA, but the production was very low. Therefore, inthis study, an enhanced GABA single-step production system using a recombinant C.glutamicum strain co-expressing two glutamate decarboxylase genes gadB1and gadB2wasestablished. It could produce GABA more effectively after the optimization of fermentationcondition. The main results are as follows.(1) Firstly, gadB1and gadB2mere expression strains ATCC13032/pDXW-10-gadB1,ATCC13032/pDXW-10-gadB2, and co-expression strain ATCC13032/pDXW-10-gadB1-gadB2were constructed. Secondly, GABA producing capacity of thethree recombinant strains was compared. ATCC13032/pDXW-10-gadB1-gadB2couldproduce4.02g/L GABA, more effectively than the other two strains. Therefore, ATCC13032/pDXW-10-gadB1-gadB2was selected for the optimization of fermentation condition.(2) Through orthogonal experimental design, the optimal seed medium was obtained(g/L): glucose25, corn steep liquor30, K2HPO4·3H2O1, urea8, MgSO40.2. The seed brothshould be precultured for8h and then inoculated into the fermentation medium.Thefermentation medium was then optimized by one-factor experiment. The optimal glucose andcorn steep liquid concentration was100g/L and4g/L, respectively. During6h to24h offermentation,2.4g/L urea was added every3.5hours. At10h of fermentation,0.1mMpyridoxal5-phosphate (PLP) was added. At last, the GABA production reached to19.64±3.77g/L and the conversion ratio of L-glutamate to GABA reached to64%.(3) The time courses during the fermentation of ATCC13032/pDXW-10-gadB1-gadB2under the optimized fermentation condition were researched. The results showed that theGAD activity was maintained during the whole fermentation and GABA was accumulatedcontinuously. Meanwhile, the pH level could be used as an indicator to reflect the GABAbiosynthesis. After prolonging the fermentation time to120h, the GABA production reachedto27.13±0.54g/L and the final conversion ratio of L-glutamate to GABA reached to74%.(4) Finally, fermentation of ATCC13032/pDXW-10-gadB1-gadB2in3L fementor wascarried out. After72h of fermentation, the GABA production reached to26g/L and theconversion ratio of L-glutamate to GABA reached to60%. GABA productivity improvedcompared to that of shake flask fermentation.